A liposome is a hollow closed vesicle consisting of a lipid bilayer formed by suspending a thin membrane of polar lipid such as a phospholipid or the like in an aqueous solution. The liposome has basically the same lipid bilayer structure as that of a biomembrane and therefore is excellent in affinity to a living body. On the basis of this characteristic, studies on an application of the liposome as a drug delivery system have become a big concern. In this application, the liposome is used as a microcapsule having an affinity to a living body. That is, various types of drugs, enzymes or the like are administered to a living body as liposome preparations in which the drugs or the like is encapsulated.
A wide variety of drugs can be encapsulated in liposomes in accordance with the administration purpose of a liposome preparation, and an example is a superoxidedismutase (superoxide disproportionation enzyme; SOD) which inactivates a superoxide anion (O.sub.2.sup.-). The superoxide anion is produced by reduction of molecular oxygen in a living body in accordance with an external stimulus by which an antigen-antibody complex is formed. Since the superoxide anion has a function of oxidatively destroying a foreign matter phagocytosized by a phagocyte, it is effective in sterilization and detoxification. If, however, active oxygen such as the superoxide anion is produced in an excessive amount, it serves as a strong factor which causes tissue lesion or inflammation, resulting in rheumatism, an inflammatory disease such as Bechet syndrome or Crohn disease, diabetes mellitus, or a cancer disease (Drug Delivery System, 2(1), 1987). Therefore, administration of the SOD which inactivates the superoxide anion is effective in the therapy of these diseases.
The SOD, however, has low stability in a living body and has only a short half-life period in blood of about six minutes. In order to eliminate these drawbacks of the SOD, various attempts have been made to encapsulate the SOD in liposomes, thereby stabilizing the SOD and allowing to slowly deliver the encapsulated SOD. For example, Published Unexamined Japanese Patent Application No. 1-175944 teaches that the SOD is held by an electrically neutral or negative film of liposome, so as to stabilize the SOD. Also Published Unexamined Japanese Patent Application No. 63-211222 teaches that a liposome film component material containing phospholipid and chloesterol is hydrated at a temperature higher than the phase transition temperature, so as to take into the inner phase the physiologically active substance which tends to be denatured or deactivated by an enzyme drug such as the SOD. Further, each of Published Unexamined Japanese Patent Application Nos. 1-238537 and 1-246225 teaches that a polypeptide having the SOD activitates is prepared so as to facilitate intake of the polypeptide into a liposome and, thus, to prepare drugs for curing liver disorders and pancreatitis.
As a membrane constituent of the liposome, a lipid, especially a phospholipid such as lecithin is used. The phospholipid generally has a hydrophilic portion consisting of a phosphate ester residue and a hydrophobic portion consisting of a higher fatty acid residue. A phospholipid having eight or less, particularly, four or less carbon atoms in this fatty acid residue portion no longer forms any molecular aggregations. Therefore, no liposome can be formed from such a phospholipid. In order to form morphologically clear liposomes from a phospholipid, therefore, the number of carbon atoms of the fatty acid residue of the phospholipid must be ten or more, and preferably, 12 to 24. Note that, in general, a lipid other than a phospholipid, for example, triglyceride, cholesterol, cholesterolester, or .alpha.-tocopherol is often added as an auxiliary for liposome formation.
For liposomes to be applied to a drug delivery system, there has been proposed a method of adding a small amount of a charged substance such as stearylamine as an assistant to a liposome so as to apply a positive electric charge to the membrane surface of the liposome. The purposes of the method are to improve a drug encapsulation ratio into the liposome and to improve the adhesion characteristics of the liposome with respect to cells. In addition, it is suggested that these cationic liposomes have better residence time in blood than those of neutral or anionic liposomes (Eur. J. Biochem., 47, 179-185 (1974)). The following two literatures can be exemplified as prior arts of this cationic liposome.
The first on is Published Unexamined Japanese Patent Application No. 63-77824 which discloses a cationic liposome medicinal preparation (L-SOD) encapsulating the SOD. In this L-SOD, stearylamine is used as a membrane constituent of the liposome in addition to dipalmitoylphosphatidylcholine and cholesterol.
The second one is "Drug Delivery System" (2(1), 41-52 (1987)) which discloses a cationic liposome medicinal preparation (L-SOD) also added with stearylamine.
Since, however, the above cationic L-SOD added with stearylamine has a serious problem of high toxicity, it causes a side effect of, e.g., convulsion when it is applied to a living body (J. Neurol. Sci., 31, 173-179 (1977)). For this reason, this cationic L-SOD has not been able to be put into practical use as a liposome medicinal preparation.